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Author(s): Karvinen, Sira; Silvennoinen, Mika; Ma, Hongqiang; Törmäkangas, Timo; Rantalainen,
Timo; Rinnankoski-Tuikka, Rita; Lensu, Sanna; Koch, Lauren G.; Britton, Steven L.;
Kainulainen, Heikki
Title:
Voluntary Running Aids to Maintain High Body Temperature in Rats Bred for High
Aerobic Capacity
Year:
2016
Version:
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Please cite the original version:
Karvinen, S., Silvennoinen, M., Ma, H., Törmäkangas, T., Rantalainen, T., RinnankoskiTuikka, R., . . . Kainulainen, H. (2016). Voluntary Running Aids to Maintain High Body
Temperature in Rats Bred for High Aerobic Capacity. Frontiers in Physiology, 7, 311.
doi:10.3389/fphys.2016.00311
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ORIGINAL RESEARCH
published: 25 July 2016
doi: 10.3389/fphys.2016.00311
Voluntary Running Aids to Maintain
High Body Temperature in Rats Bred
for High Aerobic Capacity
Sira M. Karvinen 1*, Mika Silvennoinen 1 , Hongqiang Ma 2 , Timo Törmäkangas 3 ,
Timo Rantalainen 4 , Rita Rinnankoski-Tuikka 1 , Sanna Lensu 1 , Lauren G. Koch 5 ,
Steven L. Britton 5, 6 and Heikki Kainulainen 1
1
Department of Biology of Physical Activity, Neuromuscular Research Center, University of Jyväskylä, Jyväskylä, Finland,
Department of Health Sciences, University of Jyväskylä, Jyväskylä, Finland, 3 Gerontology Research Center and Department
of Health Sciences, University of Jyväskylä, Jyväskylä, Finland, 4 Centre for Physical Activity and Nutrition Research, School of
Exercise and Nutrition Sciences, Deakin University, Melbourne, VIC, Australia, 5 Department of Anesthesiology, University of
Michigan Medical School, Ann Arbor, MI, USA, 6 Department of Molecular and Integrative Physiology, University of Michigan
Medical School, Ann Arbor, MI, USA
2
Edited by:
Igor B. Mekjavic,
Jozef Stefan Institute, Slovenia
Reviewed by:
Naoto Fujii,
University of Ottawa, Canada
Stephen Cheung,
Brock University, Canada
Kei Nagashima,
Waseda University, Japan
*Correspondence:
Sira M. Karvinen
[email protected]
Specialty section:
This article was submitted to
Exercise Physiology,
a section of the journal
Frontiers in Physiology
Received: 18 April 2016
Accepted: 07 July 2016
Published: 25 July 2016
Citation:
Karvinen SM, Silvennoinen M, Ma H,
Törmäkangas T, Rantalainen T,
Rinnankoski-Tuikka R, Lensu S,
Koch LG, Britton SL and
Kainulainen H (2016) Voluntary
Running Aids to Maintain High Body
Temperature in Rats Bred for High
Aerobic Capacity.
Front. Physiol. 7:311.
doi: 10.3389/fphys.2016.00311
Frontiers in Physiology | www.frontiersin.org
The production of heat, i.e., thermogenesis, is a significant component of the metabolic
rate, which in turn affects weight gain and health. Thermogenesis is linked to physical
activity (PA) level. However, it is not known whether intrinsic exercise capacity, aging,
and long-term voluntary running affect core body temperature. Here we use rat models
selectively bred to differ in maximal treadmill endurance running capacity (Low capacity
runners, LCR and High capacity Runners, HCR), that as adults are divergent for aerobic
exercise capacity, aging, and metabolic disease risk to study the connection between PA
and body temperature. Ten high capacity runner (HCR) and ten low capacity runner (LCR)
female rats were studied between 9 and 21 months of age. Rectal body temperature
of HCR and LCR rats was measured before and after 1-year voluntary running/control
intervention to explore the effects of aging and PA. Also, we determined whether injected
glucose and spontaneous activity affect the body temperature differently between LCR
and HCR rats at 9 vs. 21 months of age. HCRs had on average 1.3◦ C higher body
temperature than LCRs (p < 0.001). Aging decreased the body temperature level of
HCRs to similar levels with LCRs. The opportunity to run voluntarily had a significant
impact on the body temperature of HCRs (p < 0.001) allowing them to maintain body
temperature at a similar level as when at younger age. Compared to LCRs, HCRs
were spontaneously more active, had higher relative gastrocnemius muscle mass and
higher UCP2, PGC-1α, cyt c, and OXPHOS levels in the skeletal muscle (p < 0.050).
These results suggest that higher PA level together with greater relative muscle mass
and higher mitochondrial content/function contribute to the accumulation of heat in the
HCRs. Interestingly, neither aging nor voluntary training had a significant impact on core
body temperature of LCRs. However, glucose injection resulted in a lowering of the body
temperature of LCRs (p < 0.050), but not that of HCRs. In conclusion, rats born with high
intrinsic capacity for aerobic exercise and better health have higher body temperature
compared to rats born with low exercise capacity and disease risk. Voluntary running
allowed HCRs to maintain high body temperature during aging, which suggests that
high PA level was crucial in maintaining the high body temperature of HCRs.
Keywords: aerobic capacity, aging, body temperature, physical activity, skeletal muscle
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INTRODUCTION
BAT has for long been considered as the main thermogenic
organ (van den Berg et al., 2011). At present, in addition to BAT,
skeletal muscle has been demonstrated to significantly contribute
to thermogenesis (Gavini et al., 2014). Since approximately 40%
of total body mass is composed of muscle, the capacity of
skeletal muscle to contribute to whole-body energy expenditure
is reasonable. The contribution of skeletal muscle mitochondrial
uncoupling to weight gain has been demonstrated in several
mouse models (Son et al., 2004; Choi et al., 2007; Costford
et al., 2008). It has been shown that UCP3 overexpression
protects mice from high-fat diet induced insulin resistance
and obesity (Clapham et al., 2000; Choi et al., 2007). Recent
studies from genetically contrasting rat lines reveal that intrinsic
aerobic capacity has a major role in TEE, physical activity
level and non-exercise activity thermogenesis (NEAT; Novak
and Levine, 2007; Gavini et al., 2014). Rats selectively bred for
high aerobic endurance capacity (HCR = high-capacity runner)
are also spontaneously more active and consume more energy
compared to their counterparts that are selectively bred for
low aerobic endurance capacity (LCR = low-capacity runner;
Koch and Britton, 2001; Kivelä et al., 2010). Furthermore, HCRs
have lower risk to become obese and have longer lifespan
compared to LCRs, whereas LCRs are prone to gain excess
body weight and develop metabolic disorders (Wisloff et al.,
2005; Noland et al., 2007; Kivelä et al., 2010; Koch et al.,
2011).
Body temperature is tightly associated with metabolic rate
(Geiser, 1988; Heikens et al., 2011; Landsberg, 2012); in
fact 1◦ C rise in temperature is associated with a 10–13%
increment in oxygen consumption (Landsberg, 2012). The
elevation in temperature itself is responsible for speeding up
metabolism, since enzyme-catalyzed reactions are enhanced in
higher temperatures (Landsberg et al., 2009). We recently noticed
that besides the differences between the HCR and LCR rat lines
listed above, anesthetized HCRs seemed to have higher body
temperature compared to LCRs (unpublished observation). We
wanted to study this observaton more carefully and measured
the body temperature of HCR and LCR rats and studied
the effects of aging and voluntary running for 1-year. We
also followed the body temperature and spontaneous activity
during glucose tolerance and placebo test to assess the effect of
stress (injection) and high blood glucose. Our first hypothesis
was that HCR rats have intrinsically higher body temperature
compared to LCRs due to their higher mitochondrial capacity
to produce heat. Secondly, we hypothesized that aging would
decrease body temperature levels due to aging related loss of
mitochondrial function and decreased expression of the UCP
in skeletal muscle and BAT, but the difference between the
HCR/LCR rat lines would still be evident. Thirdly, we assumed
that voluntary running would increase the body temperature in
both rat lines due to increased mitochondrial biogenesis, thus
aiding to prevent age related decrease in mitochondrial function.
Our final hypothesis was that glucose injection would increase
the body temperature especially in the energy-dissipating HCRs,
since BAT as insulin sensitive tissue may have a major role in
dissipating extra energy as heat (Cannon and Nedergaard, 2004;
Stanford et al., 2013).
Thermogenesis is an energy demanding process that has a
significant contribution to daily total energy expenditure (TEE),
body weight and health (Levine et al., 1999; Rosenbaum et al.,
2008). High thermogenesis is linked to high physical activity (PA)
level, as body temperature rises as a result of increased muscular
activity (Gleeson, 1998). In rodents it has been established, that
high PA in turn is tightly associated with high aerobic capacity
(Novak et al., 2010). Whether the level of thermogenesis is due
to inborn aerobic capacity or a consequence of PA level per-se
remains unclear.
Several features affect thermogenesis such as age, physical
activity, meals (processing nutrients), stress, and in females the
stage of estrous cycle (Landsberg et al., 1984; Horan et al., 1988;
Kent et al., 1991; Yamashita et al., 1994; Kontani et al., 2002;
Waters et al., 2010). Physical activity, whether it is endurance
or strength training, or just normal daily activities, correlates
positively with body temperature (Nozu et al., 1992; Tonkonogi
et al., 2000). Thermic effect of food is known to increase
energy expenditure and thermogenesis (Rothwell and Stock,
1979; Cannon and Nedergaard, 2004), whereas stress causes
an increase both in blood glucose concentration and body
temperature via stress hormones and stress related behavior
(Blanchard et al., 2001; Vachon and Moreau, 2001). In females,
the stage of estrous cycle is known to affect the body temperature;
temperature increases during proestrus, when progesterone and
estrogen concentrations are highest, while during estrus there is
a drop in body temperature (Marrone et al., 1976; Kent et al.,
1991).
At cellular level, the amount and efficiency of mitochondria
and uncoupling proteins (UCP) play the main role in
thermogenesis (Cannon and Nedergaard, 2004; Rousset et al.,
2004). UCP1 is primarily found in the mitochondria of brown
adipose tissue (BAT), whereas UCP3 is the main uncoupling
protein expressed in skeletal muscle (Rousset et al., 2004). UCP2
is a mitochondrial uncoupling protein that separates oxidative
phosphorylation from ATP synthesis with energy dissipated as
heat. Although UCP3 and UCP2 share similar features, UCP2
is mainly thought to control the production of mitochondriaderived reactive oxygen species, whereas UCP3 plays a main
role in non-shivering thermogenesis (Boss et al., 1997; Rousset
et al., 2004). Besides UCPs, also amount and efficiency of
mitochondria to produce heat affects body temperature. PGC1α plays a key role in mitochondrial biogenesis whereas
cytochrome c (cyt c) is a crucial component of the electron
transport chain in mitochondria and hence these proteins
are essential for muscle oxidative capacity (Huttemann et al.,
2011; Lin et al., 2015). Endurance exercise is known to
increase PGC-1α levels and mitochondrial biogenesis in muscle,
although UCP levels are found to be reduced (Nozu et al.,
1992; Freyssenet et al., 1996; Pilegaard et al., 2000; Jones
et al., 2003; Russell et al., 2003; Holloszy, 2008). Aging in
general causes decrease in thermogenesis via lower UCP levels,
reduced response to noradrenaline and diminished BAT content
(Horan et al., 1988; Yamashita et al., 1994; Kontani et al.,
2002).
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MATERIALS AND METHODS
Measurement was repeated in separate days and the average of
two measurements was used for the statistical analyses.
Animal Lines
The HCR/LCR contrasting rat model was produced via twoway artificial selection, starting from a founder population of
186 genetically heterogeneous rats (N:NIH stock), as described
previously (Koch and Britton, 2001). Endurance running
capacity was assessed at the University of Michigan (Ann Arbor,
Michigan, USA) with a speed-ramped treadmill running test
(15◦ slope, initial velocity of 10 m•min−1 , increased 1 m/min
every 2 min) when the rats were 11 weeks of age. For the
glucose and placebo tests described here, 20 female rats (10
HCR and 10 LCR) from the 27th generation of selection were
used (Figure 1, Set 1). First non-trained animals were tested
before 1-year voluntary intervention (age 9 months) and the
tests were repeated as follow-up after the intervention (age
21 months). We also used tissue samples collected from 60
female rats from generations 23–27 of selection given the
same voluntary running intervention to prepare Western blots
from gastrocnemius muscle and BAT at the same time points
(Figure 1, Set 2). An additional set of 4–6 months old female
HCR/LCR rats from the generation 35 were used to measure
body surface temperatures. The rats lived in an environmentally
controlled facility (12/12 h light-dark cycle, 22◦ C) and received
water and standard feed (R36, Labfor, Stockholm, Sweden) ad
libitum.
Surface Temperature from Back and Tail
An infrared camera (FLIR A300) with thermal sensitivity of
<0.05◦ C was used to assess surface temperatures of backs
and tails of HCR/LCR rats from the generation 35 (n = 9–
12/group). For the measurement, each rat was lifted from the
home cage to a separate plastic cage and three infrared images
were taken at 5 s intervals. Using the software of the camera
manufacturer (ThermaCAM Researcher Pro 2.1.) the highest
surface temperature of the rat back and tail from each image was
recorded and the mean values were regarded as the actual back or
tail surface temperatures.
Intervention
Before the first measurements, at the age of 9 months, the rats
were housed two per cage in standard cages. After the first
measurements rats were divided into groups evenly matched
for body weight and maximal running capacity (n = 5);
HCR (control), HCR-R (runner), LCR (control) and LCR-R
(runner; Figure 1, Set 1). Control rats lived in a standard cage
conditions and runners had an access to running wheels that
were connected to a computerized recording system to follow
the running distance throughout intervention. During this 1-year
intervention the rats were housed one per cage. The follow-up
measurements were performed at the age of 21 months.
Baseline Measurements
Voluntary Running Distance, Body Weight, and
Energy Intake
Body Temperature
Body temperature was assessed by measuring rectal temperature
(Fluke 52 k/J Thermometer) after 2 h of fasting before dividing
the rats into separate intervention groups (n = 10/group).
Voluntary running distance in the running wheels was followed
throughout the 1-year intervention with a self-constructed
FIGURE 1 | Schematic representation of the study protocol. There were two sets of rats with the same 1-year intervention with 4 sub-groups: HCR (control),
HCR-R (runner), LCR (control), and LCR-R (runner). For set1 (20 rats) glucose tolerance and placebo tests were performed before the intervention (age 9 months) and
after the intervention (age 21 months). From set 2 (60 rats) gastrocnemius muscle and brown fat samples were collected from the same time points.
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Spontaneous activity
computerized recording system (Acer Verinton 6900Pro, 32 bit
processor produced by Intel, Windows XP). Total wheel laps were
recorded continuously, and the total running distance per day
was determined by multiplying the number of wheel rotations by
the circumference of the running wheel (Ø 34.5 cm). From daily
running distances an average daily running distance for every
2-week period was calculated. Body weight and energy intake
of the rats were followed throughout the 1-year intervention
by weighing the rats and consumed feed every second week.
Energy intake was calculated as 2 week feed consumption
from the energy content informed by the manufacturer
(Labfor).
Spontaneous activity of the rats was followed throughout the
glucose tolerance and placebo tests with ground reaction force
recording as described before (Silvennoinen et al., 2014). The
rat cages were placed on top of individual ground reaction force
plates 30 min before the start of the experiments and removed
from the plate after the last blood glucose measurement. The
absolute values of the differences between consecutive force
values were calculated. The mean of the absolute values were
calculated from every second from total 20 values per second.
To obtain activity index, a single value for total spontaneous
activity, the one second means were summed for the total
measurement time and the sum was divided by the body mass
(kg) of the measured rat (Biesiadecki et al., 1999; Silvennoinen
et al., 2014). The data used in statistical analyses were presented
as 30 min averages. The total spontaneous activity was also
calculated during the tests as a sum of activity index from the
whole test period.
Body Temperature: Before and After Intervention
Measurements
To study the effects of rat line, aging and voluntary running
before and after 1-year voluntary running intervention,
body temperature was measured similarly to the baseline
measurements described above. Measurements were done after
5 h of fasting from the same rats as used for the intervention
(n = 5/group). The average of the fasting (F) point measurements
from each rat from glucose tolerance and placebo tests were used
for the statistical analyses.
Blood glucose concentration
Blood glucose was measured at time points 0, 30, 60, and 120 min
after the injection (HemoCue Glucose 201 RT). AUC normalized
with 0 min concentration from the placebo and glucose tests were
used for the statistical analyses of the effect of rat line, running
and treatment on blood glucose concentration.
Glucose Tolerance and Placebo Tests
The glucose tolerance and placebo tests were performed twice,
first at the age of 9 months and second time at the age of 21
months (Figure 1). Tests were done in two parallel sets in each
day, between 9.30 a.m. and 3.30 p.m. Rats from different groups
were randomized to treatment sets to account the possible effect
of circadian rhythm on blood glucose level. Two animals were
excluded from the follow-up measurement of old rats due to
aging symptoms, leaving the following group sizes for second
measurements; HCR (n = 4), HCR-R (n = 5), LCR (n = 4)
and LCR-R (n = 5). Rats were deprived of food for 5 h before
measurements. The running wheels of the runner rats were
blocked 5 h before the measurements disabling the movement
of the wheel to avoid the possible acute effects of running on
the measurements. Body weight was measured before each test.
At the time point 0 either 2 g/kg of glucose (20% solution)
or equal volume of placebo (physiological saline solution) was
injected into the peritoneal cavity. Rectal temperature (Fluke
52 k/J Thermometer) and blood glucose were measured at
time points 0, 30, 60, and 120 min after the injection. Blood
samples for further analyses were collected from saphenous
vein before starting the glucose/placebo protocol (fasting
sample).
Phase of estrous cycle
Phase of estrous cycle was followed 1 week during the glucose
tolerance and placebo measurements after the intervention (age
21 months). Estrous cycle samples were collected as epithelium
samples with physiological saline solution from vagina to
a pipette, stained with methylene blue (Giemsas azur-eosin;
methylenblaulösung, Merck 9204) and observed under a light
microscope. Each sample was categorized to one of the four
menstrual cycle states: proestrus, estrus, diestrus, or metestrus.
Tissue Processing
Gastrocnemius muscle (n = 10/group) and BAT (n = 5/group)
samples were collected from the Set 2 rats (Figure 1). The
snap frozen samples were homogenized in liquid nitrogen
and dissolved in ice-cold buffer (20 mM HEPES (pH
7.4), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2 , 100 mM,
β-glycerophosphate, 1 mM Na3 VO4 , 2 mM DTT, 1% NP-40,
0.2% sodium deoxycholate, and 3% protease and phosphatase
inhibitor coctail (P 78443; Pierce, Rockford, IL). The muscle
homogenate was thereafter centrifuged at 10 000 g for 10
min at 4◦ C. Total protein content was determined using
the bicinchoninic acid protein assay (Pierce Biotechnology,
Rockford, IL) with an automated KoneLab instrument (Thermo
Scientific, Vantaa, Finland).
Heat accumulation
Rectal temperature was measured at time points 0, 30, 60,
and 120 min after the injection. To compare the changes in
temperature during placebo and glucose tolerance tests the area
under curve (AUC) values of rectal temperature were calculated
from each group during both tests between time points 0–120
min normalized with 0 min level. AUC values were used for the
statistical analyses of the effect of rat line, running and treatment
on heat accumulation.
Frontiers in Physiology | www.frontiersin.org
Citrate synthase activity
Citrate synthase activity (U•µg−1 •min−1 ) in the gastrocnemius
muscle (n = 10/group) was measured from the same muscle
homogenates that were used to determine the total protein
content (Citrate Synthase Assay Kit Sigma-Aldrich) with an
automated KoneLab instrument (Thermo Scientific).
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Western blot analyses
parameter of interest. Levene’s test was conducted to assess the
homogeneity of variance assumption. Univariate analysis was
done to analyze the line, age, running and treatment effects to
the measured parameters with Tukey Post-hoc test. Body surface
temperatures were analyzed using T-test. When the normality
or equality of variance assumptions were not met, statistical
comparisons of parameters between LCR and HCR groups were
made using Mann–Whitney test. The comparison between before
and after intervention parameters within the same group were
done using Wilcoxon test. Mixed model analysis controlled for
age, running (yes/no), treatment (placebo/glucose), time point
(F, 0, 30, 60, and 120 min), and spontaneous activity, was used
to determine the effect of line (HCR or LCR) on the measured
body temperature levels. Separate analysis for both rat lines of
the effect of running, treatment, time point, and spontaneous
activity on rectal temperature were performed using 4 × 5
longitudinal covariance structure analyses in Mplus 7 for both
glucose tolerance and placebo tests. P-values less than 0.05 were
considered statistically significant.
Aliquots of muscle homogenate were solubilized in Laemmli
sample buffer and heated at 95◦ C to denaturate proteins, except
for Total OXPHOS Cocktail, when samples were heated at
50◦ C. Thereafter, samples containing 30 µg of total protein
were separated by SDS-PAGE for 60 to 90 min at 200 V
using 4–20% gradient gels on Criterion electrophoresis cell (BioRad Laboratories, Richmond, CA). Proteins were transferred
to PVDF membranes at 300 mA constant current for 2 h on
ice at 4◦ C. The homogeneity of protein loading was checked
by staining the membrane with Ponceau S. Membranes were
blocked in TBS with 0.1% Tween 20 (TBS-T) containing 5%
non-fat dry milk for 2 h and then incubated overnight at 4◦ C
with commercially available polyclonal primary phosphospecific
antibodies to measure the following protein contents with stated
dilutions: GAPDH (1:10000; ab9485, Abcam), tubulin (1:1500;
T6199, Sigma), PGC-1α (1:4000; 516557, Calbiochem), cyt c
(1:500; sc-8385, Santa Cruz biotechnology, Inc.), UCP2 (1:300;
ab67241 Abcam), UCP3 (1:3000; ab3477 Abcam), and Total
OXPHOS Cocktail (1:1000; ab110413; Abcam). All the antibodies
were diluted in TBS-T containing 2.5% non-fat dry milk.
BAT samples were treated as above and prepared for Westernblot analyses for PGC-1α, cyt c, UCP1 (1:4000; ab10983, Abcam),
UCP2, and UCP3.
After the primary antibody incubation membranes were
washed in TBS-T, incubated with suitable secondary antibody
diluted in TBS-T with 2.5% milk for 1 h followed by washing
in TBS-T. Proteins were visualized by ECL according to the
manufacturer’s protocol (SuperSignal West femto maximum
sensitivity substrate, Pierce Biotechnology) and quantified using
ChemiDoc XRS in combination with Quantity One software
(version 4.6.3. Bio-Rad Laboratories). The UCP3 and pACC
membranes described above were incubated in Restore Western
blot stripping buffer (Pierce Biotechnology) for 30 min and
reprobed with GAPDH or total-ACC antibodies by immunoblot
analysis as described above. Results from gastrocnemius muscle
were normalized to the corresponding level of GAPDH (PGC1α, cyt c and UCP3), tubulin (UCP2, total-ACC and pACC), or
PonceauS stained actin band (OXPHOS Cocktail). All proteins
from BAT samples were normalized to corresponding level of
tubulin.
RESULTS
Baseline Measurements
Body Temperature
HCRs had on average 1.3◦ C higher body temperature after 2 h of
fasting compared to LCRs (p < 0.001; Figure 2A).
Surface Temperature from Back and Tail
HCRs had higher surface temperature from tail compared to
LCRs (p < 0.01; Figures 2B,C). There was no difference in the
surface temperatures measured from the back (Figures 2B,C).
Intervention Measurements
Before and After 1-Year Running Intervention
Measurements
Univariate analysis showed a significant line effect on body
temperature both before and after intervention (p < 0.050),
with HCRs having higher body temperature compared to LCRs
(Figure 3). When only control groups (HCR vs. LCR) were
included in the analyses, the line effect after intervention
diminished. Aging did not have a marked impact on body
temperature, whereas running had a significant effect after
intervention (p < 0.050) showing an increase in the body
temperature levels in both rat lines (p < 0.050, Figure 3).
Also combined effect of line and running was significant after
intervention (p < 0.050). Post-hoc test showed that after the
intervention, HCRs in the runner group had higher body
temperature compared to the corresponding controls (p <
0.010), whereas there was no difference between the LCR groups
(Figure 3).
Serum Cortisol Concentration
Cortisol concentration was measured from frozen (−80◦ C)
serum samples by a kinetic photometric method with KoneLab
(Thermo Scientific).
Ethics Statement
This study was approved by the National Animal Experiment
Board, Finland (Permit number ESAVI-2010-07989/Ym-23).
Statistical Analyses
Voluntary Running Distance, Body Mass, and Energy
Intake
All values in figures are expressed as mean ± standard error of
the mean (SEM). Statistical analyses for variables were carried
out using SPSS for Windows 22 statistical software (version
22, IBM SPSS Statistics) and in Mplus 7. The Shapiro-Wilk
test was used to investigate within group normality for a given
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HCRs ran voluntarily more than LCRs, the difference being
significant at time points 10, 10.5, 12.5, and 21 months of age
(p < 0.05; Figure 4A). When comparing the body weight, LCR
rats in both groups were heavier than HCR rats during the whole
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Aerobic Capacity and Body Temperature
FIGURE 3 | Before and after intervention measurements of body
temperature. Before and after intervention measurements of body
temperature. n = 4–5/group, **p < 0.010 post-hoc test between HCR and
HCR-R. Univariate analysis of rat line and running effect before and after
1-year intervention. Values are expressed as mean ± SEM.
glucose test, and LCR control group had the largest negative
response to glucose injection (Figure 5A). Univariate analysis
showed an effect of treatment on heat accumulation (p <
0.001, Figure 5A). After the intervention treatment still had a
significant effect on heat accumulation (p < 0.010, Figure 5B)
and the effect of line was nearly significant (p = 0.064). Responses
to both placebo and glucose injections in all groups were negative
after the intervention, with both LCR groups having greater
negative response to glucose injection compared to HCR groups
(Figure 5B).
Spontaneous activity
Total activity during the test protocols are presented in
Figures 5C,D. Univariate analysis revealed that HCRs had higher
total activity during placebo and glucose tolerance tests both
before and after the 1-year voluntary running intervention
compared to LCRs (line effect p < 0.001, Figures 5C,D).
FIGURE 2 | Baseline measurements. (A) Baseline measurement of body
temperature. n = 10/group, ***p < 0.001. (B) Baseline measurement of body
surface temperature from tail and back. n = 9–12/group, **p < 0.010. Values
are expressed as mean ± SEM. (C) Infrared image of HCR and LCR rat.
Blood glucose concentration
Before the intervention the blood glucose AUC value in
all groups was negative during placebo test and positive
during glucose tolerance test (Figure 5E). Univariate analysis
showed significant treatment effects both before and after the
intervention (p < 0.050, Figures 5E,F). Post-hoc test further
revealed that LCRs in the control group had higher blood
glucose AUC concentration compared to the corresponding
runners both before and after the intervention (p < 0.050),
whereas there was no statistical difference between the
HCR groups.
follow-up period (p < 0.05; Figure 4B). There was no statistical
difference within the rat lines. LCRs in the control groups
had higher food intake compared to corresponding HCRs, the
difference being significant at time points 15 and 19.5 months
of age (p < 0.05). Both runner groups consumed more energy
compared to the corresponding control groups (Figure 4C), but
only in HCR line this difference was significant (HCR vs. HCRR, p < 0.05).
Glucose Tolerance and Placebo Tests
Heat accumulation
Serum Cortisol Concentration
Before the intervention, heat accumulation was positive when
normalized to 0-value during placebo and negative during
HCRs had higher cortisol concentration compared to LCRs both
before and after intervention (line effect, p < 0.001, Figure 6).
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FIGURE 4 | Voluntary running distance, Body mass and Food intake during 1-year intervention. (A) Voluntary running distance (m/day). HCRs had longer
voluntary running distance than LCRs, the difference being significant at time points 10, 10.5, 12.5, and 21 months of age, *p < 0.050, **p < 0.010. (B) Body
mass (g). LCR rats in both groups were heavier compared to HCR rat groups during the follow-up period (*p < 0.050). In LCRs the rats in the runner group were
(Continued)
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FIGURE 4 | Continued
leaner than control ones, whereas the opposite was true for HCRs. There were no statistical differences within the rat lines. (C) Food intake (kcal). LCRs in the control
groups had higher food intake compared to corresponding HCRs, the difference was significant at time points 15 and 19.5 months of age (p < 0.050). Runner groups
of both rat lines consumed more energy compared to the corresponding control groups, but only in HCR line this difference was significant (HCR vs. HCR-R, p <
0.050). n = 4–5/group, values are expressed as mean ± SEM.
Western-Blot Analyses
running intervention the spontaneous activity level and protocol
time point (F, 0, 30, 60, or 120 min) had the largest effect on
body temperature of HCRs (p < 0.05, Table 3), whereas in LCRs,
treatment (placebo/glucose injection) and spontaneous activity
had highest impact on body temperature (p < 0.001). After
the intervention both running and spontaneous activity had a
significant contribution to the body temperature levels of HCRs
(p < 0.05). In LCRs treatment and time point had marked impact
on body temperature after the intervention (p < 0.05).
Gastrocnemius Muscle
Univariate analysis showed a significant line effect in UCP2 and
OXPHOS (p < 0.050) with HCRs having higher protein levels
compared to LCRs (Figure 7). HCRs had also higher PGC-1α and
cyt c protein levels than LCRs (p < 0.050, reported previously;
Figure 7). Aging also increased the level of PGC-1α (p < 0.050)
and there was a tendency in increase of cyt c level with aging (p =
0.087; reported previously). There were no statistical differences
in the other studied protein contents.
DISCUSSION
BAT
Line, running or aging had no significant effect on BAT protein
contents (data not shown).
In the present study we examined the association of intrinsic
aerobic capacity, aging, voluntary running and blood glucose
concentration on body temperature in two genetically
contrasting rat lines (HCR/LCR) that widely differ for their
intrinsic (i.e., non-trained) aerobic capacity (Koch and Britton,
2001). Our findings show that at young age, in untrained state,
HCRs have higher body temperature than LCRs. We also found
that voluntary running aided HCRs to maintain high body
temperature during aging. Glucose injection lowered the body
temperature of LCRs, whereas no significant effect of blood
glucose on body temperature was found in HCRs.
As hypothesized, our study showed that untrained HCRs had
on average 1.3◦ C higher body temperature than corresponding
LCRs (Figure 2A). There was also an apparent rat line effect
when measuring the baseline body temperatures before 1-year
voluntary running intervention (Figure 2B). Since normal body
temperature range in rats is 35.9–37.5◦ C (Animal care and
use committee, John Hopkins University, Baltimore, Maryland,
USA), HCRs seem to have slightly elevated body temperature
compared to reference values. Previous findings have shown that
female HCRs have higher muscle heat dissipation during activity,
explaining their higher total energy expenditure compared to
LCRs (Gavini et al., 2014). However, in that study critical
factor was activity related thermogenesis, whereas no significant
contribution of resting metabolic rate was found when body
size and composition were considered (Gavini et al., 2014).
As shown in previous study by Novak et al. and here, HCRs
are spontaneously more active than LCRs (Figures 5C,D) and
activity level also contributed to body temperature level (Table 3),
making spontaneous activity a potential cause for the higher
thermogenesis of HCRs (Novak et al., 2010). However, the total
activity of the rats was very similar during both placebo and
glucose tolerance test showing no effect of treatment on activity
level (Figures 5C,D). Yet there was a clear difference in the
heat accumulation between the two protocols (Figures 4A,B),
indicating that the higher heat accumulation of HCRs is not
solely explained by their PA level. Although the back surface
Citrate Synthase Activity
HCRs had higher citrate synthase activity compared to LCRs
(line effect p < 0.05, reported previously). The activity levels were
following: Before intervention: HCR 2939 ± 679, LCR 2767 ±
866 and after intervention: HCR 3267 ± 1081, HCR-R 3554 ±
835, LCR 2863 ± 917 and LCR-R 3545 ± 1328 (U•µg−1 •min−1 ,
mean ± SD). Aging or voluntary running had no significant effect
on citrate synthase activity.
Body Mass and Relative Gastrocnemius
Muscle and Bat Masses
Body and relative masses of gastrocnemius muscle and BAT
are listed in Table 1. Before the intervention, HCRs had lower
body mass and higher relative gastrocnemius muscle mass
compared to LCRs (line effect p < 0.001). Aging had a significant
effect in both body mass and relative gastrocnemius muscle
mass (p < 0.050); before the intervention HCRs had higher
relative gastrocnemius muscle mass compared to HCRs after the
intervention, while LCRs before intervention had higher relative
gastrocnemius mass compared to both LCR and LCR-R after
intervention. Line, running or age had no significant effect on
relative BAT mass.
Estrous Cycle
There were no statistical differences between the groups in the
observed stages of estrous cycle during glucose tolerance and
placebo tests (Table 2).
Mixed Model and Longitudinal Covariance
Structure Analysis of Body Temperature
Mixed model analyses from intervention measurements revealed,
that rat line had clear impact on body temperature both
before and after intervention (p < 0.01, Table 3). Longitudinal
covariance analysis showed that before the 1-year voluntary
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FIGURE 5 | Heat accumalation (AUC rectal temperature), total activity and blood glucose concentration (AUC) during placebo and glucose tolerance
tests. (A,B) AUC Rectal temperature. Before the 1-year intervention treatment had a significant effect on heat accumulation (p < 0.001), with glucose injection
lowering the heat accumulation. After the intervention treatment was still significant (p < 0.010) and line effect was nearly significant (p = 0.064). (C,D) Total activity.
HCRs had higher total activity compared to LCRs both before and after 1-year intervention (line effect p < 0.001). (E,F) AUC Glucose concentration. Treatment had a
significant contribution to blood glucose AUC concentration both before and after intervention (p < 0.001), with glucose injection increasing blood glucose AUC.
Post-hoc test revealed that LCRs controls had higher blood glucose AUC compared to the corresponding runners both before and after the intervention (*p < 0.050).
Univariate analysis of effects of rat line, treatment (glucose/placebo injection) and voluntary running on studied parameters. n = 4–5/group, values are expressed as
mean ± SEM.
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tissue level, aging did diminish the difference in the body
temperature between the rat lines when comparing only the
control groups (HCR vs. LCR, Figure 2B). This was due to
decrease in body temperature of HCRs; aging did not have
a marked impact on the body temperature of LCRs. For an
unknown reason, HCRs seem to lose their ability to keep up high
heat generation with aging at control conditions (e.g., no running
wheel). Our results revealed that aging significantly diminished
the relative gastrocnemius muscle mass in HCRs (Table 1), which
may partly contribute to the aging related lower heat generation.
As we hypothesized, 1-year voluntary running did increase the
body temperature in both rat lines showing a significant running
effect (Figure 3). Further analyses showed that running had a
significant impact on the body temperature of HCRs (Table 3),
which is consistent with their running distances compared to
LCRs (Figure 4A). It has been established in previous studies,
that endurance training increases PGC-1α and cyt c levels as
well as mitochondrial content and respiratory capacity in skeletal
muscle in both humans and rodents (Holloszy and Coyle, 1984;
Booth, 1991; Baar et al., 2002; Pilegaard et al., 2003). Surprisingly,
voluntary running had no significant effect on the mitochondrial
proteins in the present study. It seems that in gastrocnemius
muscle, there was no pressure to increase the mitochondrial
proteins at time point chosen (age of 21 months). PGC-1α is
known to respond to exercise acutely (Baar et al., 2002). Like in
other studies, also in our study the running distance decreased
gradually over time (Holloszy, 1993; Judge et al., 2005), also
the pressure for change decreased (Figure 4A). Contrary to the
increased body temperature by voluntary running established
in our study, in previous studies endurance training has been
associated with decreased mRNA expression of the uncoupling
proteins in skeletal muscle and reduced thermogenesis in BAT
(Nozu et al., 1992; Boss et al., 1998).
It is worth noting that lifespan is generally negatively
correlated with body temperature (Rikke and Johnson, 2004).
Several life-extending manipulations in rodents, such as caloric
restriction, have shown to decrease body temperature by
1–5◦ C (Rikke and Johnson, 2004). Furthermore, a modest
prolonged reduction of core body temperature (0.3–0.5◦ C)
increased median life expectancy in mice 12–20% even without
caloric restriction (Conti et al., 2006). Nonetheless, in the
HCR/LCR animal model, HCRs have higher body temperature
associated with longer lifespan than LCRs (Koch et al.,
2012; Karvinen et al., 2015). The free radical hypothesis
of aging includes ‘uncoupling to survive’ hypothesis, which
suggests that correlation between metabolic rate and longevity
should be positive (Brand, 2000). In a previous study, it
was estimated that mitochondrial proton cycling causes up to
20–25% of basal metabolic rate in rats (Rolfe et al., 1999).
It was suggested that the function of the energy-dissipating
proton leak is not primarily to increase thermogenesis, but
to decrease the production of reactive oxygen species. Indeed,
proton leak is proposed to be a key factor in aiding to
decrease oxidative damage to DNA and to slow down aging
(Brand, 2000; Speakman et al., 2004). It seems that HCRs
are good candidates supporting the ‘uncoupling to survive’
hypothesis since their higher OXPHOS, PGC-1α, and UCP2
FIGURE 6 | Cortisol. HCRs had higher cortisol concentrations compared to
LCRs both before and after intervention (line effect p < 0.001). Serum analyses
were done from the fasting blood samples collected before and after
intervention. n = 4–5/group, values are expressed as mean ± SEM.
temperatures of HCRs and LCRs did not differ, HCRs had
significantly higher tail surface temperature (Figures 2B,C).
Since rats use their tail blood circulation for thermoregulation
(Raman et al., 1983), HCRs seem to dissipate more heat
suggesting that HCRs also have higher thermogenesis than LCRs.
This is also supported by our previous results showing that HCRs
have higher resting metabolic rate than LCRs (Kivelä et al., 2010).
In the present study HCRs had higher UCP2, PGC-1α, cyt
c, and OXPHOS levels compared to LCRs, showing a greater
oxidative phosphorylation capacity (Figure 7). Interestingly, a
previous study has shown that overexpression of PGC-1α in
skeletal muscle leads to elevated proton leak i.e., less efficient
mitochondrial respiration (St-Pierre et al., 2003). Since HCRs
have more OXPHOS proteins and higher UCP2 and PGC-1α
levels in skeletal muscle, it may be speculated that the higher
proton leakage may also be one reason for HCRs higher heat
production. On the other hand, several animal models have
demonstrated that rodents with inherited obesity have low body
temperatures (Trayhurn et al., 1977; Levin et al., 1981; Dubuc
et al., 1985). Here we show for the first time, that similarly
to inherited obesity, LCRs have lower body temperature than
HCRs, and are prone to gain excess weight and develop metabolic
disorders (Koch and Britton, 2005; Noland et al., 2007; Kivelä
et al., 2010).
Contrary to our second hypothesis, aging per-se had no
significant effect on body temperature (Figure 3). This is in line
with a previous study, where aging did not have an impact on
rectal temperatures (3–24 months old), until in later age (36
months; Horan et al., 1988; McDonald et al., 1989). However,
aging increased the level of PGC-1α and had a tendency to
increase cyt c level (Figure 7), not showing expected aging
related decrease of mitochondrial function and content as shown
previously (Conley et al., 2000; Huang and Hood, 2009; Johnson
et al., 2013). Despite these unexpected findings from muscle
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FIGURE 7 | Western blot analyses from gastrocnemius muscle. HCRs had higher UCP2, PGC-1α, cyt c, and OXPHOS protein levels (line effect p < 0.050).
Aging increased the level of PGC-1α (p < 0.050) and there was a tendency in increase of cyt c level with aging (p = 0.087). n = 9–10/group, values are expressed as
mean ± SEM.
rate of metabolism are beneficial to health and longevity, and
more studies are needed to investigate the role of metabolic rate
on aging and longevity.
Our final hypothesis was that glucose injection would
increase the body temperature especially in HCRs, increasing
heat generation in BAT. Our results revealed, that especially
in LCRs, heat accumulation was lower after glucose tolerance
test compared to placebo test (Figures 5A,B). Longitudinal
covariance analysis further revealed that treatment was
a significant contributor to temperature level in LCRs
both before and after intervention (Table 3). It may be
speculated, that in LCRs glucose injection activates energy
storage mechanisms that decrease their metabolic rate shortly
after energy supplementation (Ravussin and Gautier, 1999;
Almind and Kahn, 2004; Landsberg et al., 2009; Heikens
et al., 2011). However, it should be noticed that there was a
difference in the blood glucose AUC level between the LCR
groups already before the 1-year running intervention started
(Figure 5C), which is most likely due to large variation in the
response to glucose dose due to small group size. After the
intervention rats in the running groups had a lower response
to glucose; however, again the post-hoc tests revealed that
the difference was only significant between the LCR groups
(Figure 5E).
In addition to aging and voluntary running, other potential
factors may affect the body temperature in our setup, e.g.,
handling of the animal and estrous cycle. Handling is known to
have a stress effect on rats and it can cause a series of behavioral
and physiological responses, such as flight response, freezing
response, increase in heart rate, urination and increase in plasma
glucocorticoid levels (Rodgers et al., 1997; Blanchard et al., 2001).
TABLE 1 | Background information for Western blot analyses.
Body mass (g)
Gastrocnemius/Body
mass (mg/g)
HCR_young
232 ± 30
5.32 ± 0.65
HCR_old
260 ± 36
4.57 ± 0.58
1.33 ± 0.22
HCR-R old
268 ± 34
4.78 ± 0.49
1.23 ± 0.27
LCR_young
302 ± 26
4.73 ± 0.43
LCR_old
320 ± 37
4.21 ± 0.45
1.02 ± 0.36
LCR-R_old
345 ± 48
4.00 ± 0.59
1.16 ± 0.26
p
Line < 0.001***
Line < 0.001***
Age < 0.050*
Age < 0.001***
Brown fat/Body
mass (mg/g)
Body and relative tissue masses (tissue mass/body mass) of the rats used for western
blot analyses. n = 10/group, values are expressed as mean ± SD.
levels combined with higher body temperature and longer
lifespan compared to LCRs (Koch et al., 2012; Karvinen et al.,
2015).
Previous studies have also shown that core body temperature
declines with age both in rodents and in humans (Roth et al.,
2002; Sanchez-Alavez et al., 2011; Waalen and Buxbaum, 2011),
which has raised speculation of possible anti-aging effects of low
body temperature. In our studies, HCRs in the control group
had lower body temperature at the age of 21 months than HCRRs, and our previous study reported that HCRs in control group
had also longer lifespan (Karvinen et al., 2015). According to our
results it can be speculated that voluntary running may interfere
with a natural aging-related reduction in body temperature of
HCRs, possibly contributing to a decrease in lifespan. However, it
remains controversial whether high body temperature and high
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TABLE 2 | Stage of estrous cycle.
N
HCR
Placebo
HCR-R
Glucose
Placebo
LCR
Glucose
Placebo
LCR-R
Glucose
Placebo
Glucose
STAGE OF ESTROUS CYCLE
Proestrus
1
1
0
0
1
1
1
2
Estrus
0
0
0
3
1
2
1
2
Metestrus
2
1
2
1
1
1
2
1
Diestrus
1
2
3
1
1
0
1
0
Estimated stage of estrous during the second (age 21 months) glucose tolerance and placebo tests. Numbers are n of animals that are in the presented stage of estrous cycle. There
were no statistical differences between the groups.
To conclude, HCRs have higher basal body temperature than
LCRs at untrained state at young age. Voluntary running aids
older HCRs to maintain body temperature at similar levels
as untrained, younger HCRs. The elevated body temperature
itself may partly cause the heightened oxidative metabolism in
HCRs, as enzyme-catalyzed reactions are enhanced in higher
temperatures (Landsberg et al., 2009). However, voluntary
running may interfere with a natural aging-related reduction in
body temperature of HCRs, possibly contributing to a decrease
in lifespan (Karvinen et al., 2015). On the other hand it is
proposed that low body temperature is one reason for the onset
of obesity in humans (Landsberg et al., 2009). Yet it remains
controversial whether high body temperature and high rate of
metabolism are beneficial to health and longevity. However,
since there is clear evidence of obese animals having low body
temperatures (Trayhurn et al., 1977; Levin et al., 1981; Dubuc
et al., 1985), it would be worth studying the potential role of
intrinsically low body temperature at the onset of obesity in
humans.
TABLE 3 | Body temperature during test protocols: effect of measured
parameters.
Factor
Before intervention
HCR
LCR
Line
0.510
Treatment
0.067
Time point
0.036*
Spontaneous activity
0.037*
Running
Time point
HCR
LCR
< 0.000***
Running
Treatment
After intervention
p
0.955
< 0.001***
0.402
Spontaneous activity
< 0.001***
Line
< 0.010**
Running
< 0.001***
Treatment
0.815
Time point
0.387
Spontaneous activity
0.014*
Running
0.330
Treatment
0.013*
Time point
0.010*
Spontaneous activity
0.370
AUTHOR CONTRIBUTIONS
HK, MS, and SK designed the study. HK led the animal
experiment and SK performed the animal experiment
and analyzed the data. HM, MS, SK, and RT collected
the tissue samples. TR and MS built up the spontaneous
activity measurement system. TT and SL assisted with
the statistical analysis and interpretation of the data. SK,
HK, and MS drafted the manuscript. SB and LK bred and
phenotyped the animals. All authors contributed to the revision
of the manuscript and approved the final version of the
manuscript.
Mixed model and longitudinal covariance structure analysis of how measured parameters
affect absolute body temperature levels during placebo and glucose tolerance tests.
Parameters: running (yes/no), treatment (glucose/placebo injection), time point (F, 0, 30,
60, or 120 min), and spontaneous activity before and after voluntary running intervention.
n = 4–5 /group. *p < 0.050, **p < 0.010, ***p < 0.001.
It is known that HCRs have stronger response to stress, and this
can be observed also in elevated corticosterone level in blood
in HCRs compared to LCRs (Waters et al., 2010). Similarly in
our study HCRs had higher cortisol concentration compared to
LCRs the (Figure 6). This stronger response to stress can also be
one reason for the higher body temperature of HCRs (Vachon
and Moreau, 2001). Estrous cycle affects the body temperature in
female rats; it increases during proestrus, and drops during estrus
(Marrone et al., 1976; Kent et al., 1991). In our study the stage of
estrous cycle was estimated during the follow-up measurements.
Rats were randomly in one of the four estrous phases, and no
significant differences in the estrous cycle stages between the rat
lines were observed (Table 2).
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FUNDING
This study was funded by the Finnish Ministry of Education
and Culture, National Doctoral Programme of Musculoskeletal
Disorders and Biomaterials (TBDP) and Eemil Aaltonen
foundation. The LCR-HCR rat model system was funded
by the Office of Research Infrastructure Programs/OD grant
P40OD021331 (to LK and SB) from the National Institutes of
Health.
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Aerobic Capacity and Body Temperature
ACKNOWLEDGMENTS
tests. We are grateful to Juha Hulmi for his excellent guidance
in making the muscle homogenates and running the Western
blots, and Aino Poikonen and Juho Hyödynmaa for their help
in the Western blot analyses. We acknowledge the expert care of
the rat colony provided by Molly Kalahar and Lori Heckenkamp.
Contact LK ([email protected]) or SB ([email protected]) for
information on the LCR and HCR rats: these rat models are
maintained as an international resource with support from the
Department of Anesthesiology at the University of Michigan,
Ann Arbor, Michigan.
We thank Eliisa Kiukkanen and Laura Pitkänen (University of
Jyväskylä, Finland) for the excellent animal care. We are deeply
grateful to Mervi Matero for her help during the measurements
and dedication to the wellbeing of the animals. We are grateful
to Leena Tulla for her crucial help in collecting and organizing
the samples. We also want to thank Risto Puurtinen and Aila
Ollikainen for their help in blood analyses and Maria Mikkonen
for her skillful help in the first glucose tolerance and placebo
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2016 Karvinen, Silvennoinen, Ma, Törmäkangas, Rantalainen,
Rinnankoski-Tuikka, Lensu, Koch, Britton and Kainulainen. This is an open-access
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